The Silmarillion (Quenya: [silmaˈriliɔn]) is a collection of mythopoeic works by English writer J. R. R. Tolkien, edited and published posthumously by his son, Christopher Tolkien, in 1977, with assistance from Guy Gavriel Kay. The Silmarillion, along with J. R. R. Tolkien's other works, forms an extensive, though incomplete, narrative that describes the universe of Eä in which are found the lands of Valinor, Beleriand, Númenor, and Middle-earth, within which The Hobbit and The Lord of the Rings take place.
1977 George Allen & Unwin hardback edition. The cover features Tolkien's drawing of an heraldic emblem for Lúthien.
After the success of The Hobbit, Tolkien's publisher requested a sequel. Tolkien sent them an early draft of The Silmarillion but they rejected the work as being obscure and "too Celtic". The result was that Tolkien began work on "A Long Expected Party", the first chapter of what he described at the time as "a new story about Hobbits", which became The Lord of the Rings.
The Silmarillion comprises five parts. The first part, Ainulindalë, tells of the creation of Eä, the "world that is". Valaquenta, the second part, gives a description of the Valar and Maiar, the supernatural powers in Eä. The next section, Quenta Silmarillion, which forms the bulk of the collection, chronicles the history of the events before and during the First Age, including the wars over the Silmarils that gave the book its title. The fourth part, Akallabêth, relates the history of the Downfall of Númenor and its people, which takes place in the Second Age. The final part, Of the Rings of Power and the Third Age, is a brief account of the circumstances which led to and were presented in The Lord of the Rings.
The five parts were initially separate works, but it was the elder Tolkien's express wish that they be published together. Because J. R. R. Tolkien died before he finished revising the various legends, Christopher gathered material from his father's older writings to fill out the book. In a few cases, this meant that he had to devise completely new material in order to resolve gaps and inconsistencies in the narrative.
The Silmarillion, like Tolkien's other Middle-earth writings, was meant to have taken place at some time in Earth's past. In keeping with this idea, The Silmarillion is meant to have been translated from Bilbo's three-volume Translations from the Elvish, which he wrote while at Rivendell.
The inside title page contains an inscription written in Tengwar. In the English it reads "The tales of the First Age when Morgoth dwelt in Middle-earth and the Elves made war upon him for the recovery of the Silmarils to which are appended the downfall of Númenor and the history of the Rings of Power and the Third Age in which these tales come to their end."
The first section of The Silmarillion, Ainulindalë ("The Music of the Ainur"), takes the form of a primary creation narrative. Eru ("The One"), also called Ilúvatar ("Father of All"), first created the Ainur, a group of eternal spirits or demiurges, called "the offspring of his thought". Ilúvatar brought the Ainur together and showed them a theme, from which he bade them make a great music. Melkor – whom Ilúvatar had given the "greatest power and knowledge" of all the Ainur – broke from the harmony of the music to develop his own song. Some Ainur joined him, while others continued to follow Ilúvatar, causing discord in the music. This happened thrice, with Eru Ilúvatar successfully overpowering his rebellious subordinate with a new theme each time. Ilúvatar then stopped the music and showed them a vision of Arda and its peoples. The vision disappeared after a while, and Ilúvatar offered the Ainur a chance to enter into Arda and govern over the new world.
Many Ainur descended, taking physical form and becoming bound to that world. The greater Ainur became known as the Valar, while the lesser Ainur were called the Maiar. The Valar attempted to prepare the world for the coming inhabitants (Elves and Men), while Melkor, who wanted Arda for himself, repeatedly destroyed their work; this went on for thousands of years until, through waves of destruction and creation, the world took shape.
Valaquenta ("Account of the Valar") describes Melkor and each of the 14 Valar in detail, as well as a few of the Maiar. It also reveals how Melkor seduced many Maiar – including those who would eventually become Sauron and the Balrogs – into his service.
Quenta Silmarillion ("The History of the Silmarils"), which makes up the bulk of the book, is a series of interconnected tales set in the First Age that make up the tragic saga of the three jewels, the Silmarils.
The Valar had attempted to fashion the world for Elves and Men, but Melkor continually destroyed their handiwork. After he destroyed the two lamps that illuminated the world, the Valar moved to Aman, a continent to the west of Middle-earth, where they established their home called Valinor. Yavanna created Two Trees that illuminated Valinor and left Middle-earth to darkness and Melkor. Soon after, stars created by Varda began to shine and the Elves awoke. The elves originally formed three groups: the Vanyar, the Noldor, and the Teleri, though some elves were captured and enslaved by Melkor, eventually to be bred into orcs. Knowing the danger the Elves were in, the Valar decided to fight Melkor to keep the Elves safe. After defeating and capturing Melkor, they invited the Elves to live in Aman. Many Elves travelled to Aman, while others refused and still others stopped along the way, including the Elves who later became the Sindar, ruled by the Elf King Thingol and Melian, a Maia. Of the three tribes that set out, all of the Vanyar and Noldor, and many of the Teleri reached Aman.
In Aman, Fëanor, son of Finwë, King of the Noldor, created the Silmarils, jewels that glowed with the light of the Two Trees. Melkor, who had been held in captivity by the Valar, was eventually released after feigning repentance. Upon release, Melkor deceived Fëanor into believing that his eldest half-brother Fingolfin was attempting a coup against Finwë. This rift led to the banishment of Fëanor from the Noldor city Tirion, and Fëanor thereafter created the fortress Formenos to the North of Tirion. Finwë moved to Formenos to live with Fëanor, his favourite of three sons, and there they safely kept the Silmarils. After many years, Fëanor travelled to Tirion to make amends with Fingolfin, while Finwë stayed at Formenos. While making amends, Melkor destroyed the Two Trees with the help of Ungoliant, a dark spider spirit Melkor found in Avathar, on the Southeast side of Aman. Together, Melkor and Ungoliant then escaped to Formenos, killed Finwë, stole the Silmarils, and fled to Middle-earth. Melkor kept the Silmarils and banished Ungoliant, later attacking the Elvish kingdom of Doriath, ruled by the Elf King Thingol and his wife Melian. He was defeated in the first of five battles of Beleriand, however, and barricaded himself in his northern fortress of Angband.
Fëanor and his sons swore an oath of vengeance against Melkor – and against anyone who withheld the Silmarils from them, even the Valar. Fëanor persuaded most of the Noldor to pursue Melkor, whom Fëanor renamed as Morgoth, into Middle-earth. Fëanor's sons seized ships from the Teleri, attacking and killing many of them, and left the other Noldor to make the voyage by foot. Upon arriving in Middle-earth, the Noldor under Fëanor attacked Melkor and defeated his host, though Fëanor was slain by Balrogs. After a period of peace, Melkor attacked the Noldor but was again defeated and besieged. Nearly 400 years later, he broke the siege and drove the Noldor back.
After the destruction of the Trees and the theft of the Silmarils, the Valar created the moon and the sun. At the same time, Men awoke, some of whom later arrived in Beleriand and allied themselves with the Elves. Beren, a man who had survived the latest battle, wandered to Doriath, where he fell in love with the elf Lúthien, the king's daughter. The king sought to prevent their marriage by imposing what he believed an impossible task: retrieving one of the Silmarils from Melkor. But together, Beren and Lúthien embarked on this quest. Sauron, a powerful servant of Melkor, imprisoned Beren along the way; but with Lúthien's help he escaped, crept into Melkor's fortress, and stole a Silmaril from Melkor's crown. Having achieved the task, the first union of man and elf was formed, though Beren was soon mortally wounded and Lúthien also died of grief. However she convinced the Vala Mandos to revive Beren and herself, choosing to sacrifice her immortality in favour of a mortal life with her beloved.
The Noldor, seeing that a mortal and an elf-woman could infiltrate Angband, perceived that Melkor was not invincible. They attacked again with a great army of Elves, Dwarves and Men. But they were deceived by Melkor, who had secretly darkened the hearts of many of the men. Thus it was that the Elvish host were utterly defeated, due in part to the treachery of some Men. However, many Men remained loyal to the Elves and were honoured thereafter.
None received more honour than the brothers Húrin and Huor. Huor died in battle, but Melkor captured Húrin, and cursed him to watch the downfall of his kin. Húrin's son, Túrin Turambar, was sent to Doriath, leaving his mother and unborn sister behind in his father's kingdom (which had been overrun by the enemy). Túrin achieved many great deeds of valour, the greatest being the defeat of the dragon Glaurung. Despite his heroism, however, Túrin was plagued by the curse of Melkor, which led him unwittingly to murder his friend Beleg and to marry and impregnate his sister Nienor, whom he had never met before, and who had lost her memory through Glaurung's enchantment. Before their child was born, the bewitchment was lifted as the dragon lay dying. Nienor, realizing what grew within her, took her own life. Upon learning the truth, Túrin threw himself on his sword.
Huor's son, Tuor, became involved in the fate of the hidden Noldorin kingdom of Gondolin. He married the elf Idril, daughter of Turgon, Lord of Gondolin (the second union between Elves and Men). When Gondolin fell, betrayed from within by Maeglin, Tuor saved many of its inhabitants from destruction. All of the Elvish kingdoms in Beleriand eventually fell, and the refugees fled to a haven by the sea created by Tuor. The son of Tuor and Idril, Eärendil the Half-elven, was betrothed to Elwing, herself descended from Beren and Lúthien. Elwing brought Eärendil the Silmaril of Beren and Lúthien, and using its light Eärendil travelled across the sea to Aman to seek help from the Valar. The Valar obliged; they attacked and defeated Melkor, completely destroying his fortress Angband and sinking most of Beleriand; and they expelled Melkor from Arda. This ended the First Age of Middle-earth. The last two Silmarils were taken by Fëanor's surviving sons Maedhros and Maglor. However, Maedhros killed himself by leaping into a fiery chasm with a Silmaril while Maglor threw his into the sea and spent the rest of his days wandering along the shores of the world, singing his grief.
Eärendil and Elwing had two children: Elrond and Elros. As descendants of immortal elves and mortal men, they were given the choice of which lineage to belong to: Elrond chose to belong to the Elves, his brother to Men. Elros became the first king of Númenor and lived to 500 years old, being given an extended life in return for choosing to be a Man.
Akallabêth ("The Downfallen") comprises about 30 pages, and recounts the rise and fall of the island kingdom of Númenor, inhabited by the Dúnedain. After the defeat of Melkor, the Valar gave the island to the three loyal houses of Men who had aided the Elves in the war against him. Through the favour of the Valar, the Dúnedain were granted wisdom and power and life more enduring than any other of mortal race had possessed, making them comparable to the High-Elves of Aman. Indeed, the isle of Númenor lay closer to Aman than to Middle-earth. But their power lay in their bliss and their acceptance of mortality. The fall of Númenor came about in large measure through the influence of the corrupted Maia Sauron (the chief servant of Melkor), who arose during the Second Age and tried to conquer Middle-earth.
The Númenóreans moved against Sauron, who saw that he could not defeat them by force and allowed himself to be taken as a prisoner to Númenor. There he quickly enthralled the king, Ar-Pharazôn, urging him to seek out the immortality that the Valar had apparently denied him, thus nurturing the seeds of envy that the Númenóreans had begun to hold against the Elves of the West and the Valar. So it was that all the knowledge and power of Númenor was turned towards seeking an avoidance of death; but this only weakened them and sped the gradual waning of the lifespans to something more similar to that of other Men. Sauron urged them to wage war against the Valar themselves to win immortality, and to worship his master Melkor, whom he said could grant them their wish. Ar-Pharazôn created the mightiest army and fleet Númenor had seen, and sailed against Aman. The Valar and Elves of Aman, stricken with grief over their betrayal, called on Ilúvatar for help. When Ar-Pharazôn landed, Ilúvatar destroyed his fleet and drowned Númenor itself as punishment for the rebellion against the rightful rule of the Valar. Ilúvatar created a great wave, such as had never before been seen, which utterly destroyed and submerged the isle of Númenor, killing all but those Dúnedain who had already sailed east, and changing the shape of all the lands of Middle-earth.
Sauron's physical manifestation was also destroyed in the ruin of Númenor, but as a Maia, his spirit returned to Middle-earth, no longer able to take upon himself the fair form he once had. Some Númenóreans who had remained loyal to the Valar were spared and were washed up on the shores of Middle-earth. Among these survivors were Elendil their leader, a descendant of Elros, and his two sons Isildur and Anárion who had also saved a seedling from Númenor's white tree, the ancestor of that of Gondor. They founded the Númenórean Kingdoms in Exile: Arnor in the north and Gondor in the south. Elendil reigned as High-king of both kingdoms, but committed the rule of Gondor jointly to Isildur and Anárion. The power of the kingdoms in exile was greatly diminished from that of Númenor, "yet very great it seemed to the wild men of Middle-earth".
At the end, it is mentioned that the sunken Númenor came to be called "Atalantë", a name not used when it existed. This led many readers to the conclusion that Númenor is Atlantis; this direct link was, however, denied by Tolkien himself, who asserted that it's a natural word following the constructs of Quenya.
The concluding section of the book, comprising about 20 pages, describes the events that take place in Middle-earth during the Second and Third Ages. In the Second Age, Sauron emerged as the main power in Middle-earth, and the Rings of Power were forged by Elves led by Celebrimbor. Sauron secretly forged his own ring to control the others, which led to war between the peoples of Middle-earth and Sauron, culminating in the War of the Last Alliance, in which Elves and the remaining Númenóreans united to defeat Sauron, bringing the Second Age to an end. The Third Age began with the passing of the One Ring to Isildur, who was ambushed at the Gladden Fields shortly afterwards, and lost the ring in the River Anduin. This section also gives a brief overview of the events leading up to and taking place in The Lord of the Rings, including the waning of Gondor, the re-emergence of Sauron, the White Council, Saruman's treachery, and Sauron's final destruction along with the One Ring, after which the ages of magic end.
Tolkien first began working on the stories that would become The Silmarillion in 1914, intending them to become an English mythology that would explain the origins of English history and culture. Much of this early work was written while Tolkien, then a British officer returned from France during World War I, was in hospital and on sick leave. He completed the first story, "The Fall of Gondolin", in late 1916.
He called his collection of nascent stories The Book of Lost Tales. This became the name for the first two volumes of The History of Middle-earth, which include these early texts. The stories employ the narrative device of a mariner named Eriol (in later versions, an Anglo-Saxon named Ælfwine) who finds the island of Tol Eressëa, where the Elves live; and the Elves tell him their history. However, Tolkien never completed The Book of Lost Tales; he left it to compose the poems "The Lay of Leithian" and "The Lay of the Children of Húrin".
The first complete version of The Silmarillion was the "Sketch of the Mythology" written in 1926 (later published in Volume IV of The History of Middle-earth). The "Sketch" was a 28-page synopsis written to explain the background of the story of Túrin to R. W. Reynolds, a friend to whom Tolkien had sent several of the stories. From the "Sketch" Tolkien developed a fuller narrative version of The Silmarillion called Quenta Noldorinwa (also included in Volume IV). The Quenta Noldorinwa was the last version of The Silmarillion that Tolkien completed.
In 1937, encouraged by the success of The Hobbit, Tolkien submitted to his publisher George Allen & Unwin an incomplete but more fully developed version of The Silmarillion called Quenta Silmarillion, but they rejected the work as being obscure and "too Celtic". The publisher instead asked Tolkien to write a sequel to The Hobbit. Tolkien began to revise The Silmarillion, but soon turned to the sequel, which became The Lord of the Rings. He renewed work on The Silmarillion after completing The Lord of the Rings, and he greatly desired to publish the two works together. But when it became clear that would not be possible, Tolkien turned his full attention to preparing The Lord of the Rings for publication.
In the late 1950s Tolkien returned to The Silmarillion, but much of his writing from this time was concerned more with the theological and philosophical underpinnings of the work than with the narratives themselves. By this time, he had doubts about fundamental aspects of the work that went back to the earliest versions of the stories, and it seems that he felt the need to resolve these problems before he could produce the "final" version of The Silmarillion. During this time he wrote extensively on such topics as the nature of evil in Arda, the origin of Orcs, the customs of the Elves, the nature and means of Elvish rebirth, and the "flat" world and the story of the Sun and Moon. In any event, with one or two exceptions, he wrought little change to the narratives during the remaining years of his life.
For several years after his father's death, Christopher Tolkien compiled a Silmarillion narrative. Christopher's intentions seem to have been mostly to use the latest writings of his father's that he could, and to keep as much internal consistency (and consistency with The Lord of the Rings) as possible, though he admitted that a complete consistency was impossible. As explained in The History of Middle-earth, Christopher drew upon numerous sources for his narrative, relying on post-Lord of the Rings works where possible, but ultimately reaching back as far as the 1917 Book of Lost Tales to fill in portions of the narrative which his father had planned to write but never addressed. In one later chapter of Quenta Silmarillion, "Of the Ruin of Doriath", which had not been touched since the early 1930s, he had to construct a narrative practically from scratch. The final result, which included genealogies, maps, an index, and the first-ever released Elvish word list, was published in 1977. Because of Christopher's extensive explanations (in The History of Middle-earth) of how he compiled the published work, much of The Silmarillion has been debated by readers. Christopher's task is generally accepted as very difficult given the state of his father's texts at the time of his death: some critical texts were no longer in the Tolkien family's possession, and Christopher's task compelled him to rush through much of the material. Christopher reveals in later volumes of The History of Middle-earth many divergent ideas which do not agree with the published version. Christopher Tolkien has suggested that, had he taken more time and had access to all the texts, he might have produced a substantially different work. But he was compelled by considerable pressure and demand from his father's readers and publishers to produce something publishable as quickly as possible.
In October 1996, Christopher Tolkien commissioned illustrator Ted Nasmith to create full-page full-colour artwork for the first illustrated edition of The Silmarillion. It was published in 1998, and followed in 2004 by a second edition featuring corrections and additional artwork by Nasmith.
During the 1980s and 1990s, Christopher Tolkien published most of his father's Middle-earth writings as the 12-volume The History of Middle-earth series. In addition to the source material and earlier drafts of several portions of The Lord of the Rings, these books greatly expand on the original material published in The Silmarillion, and in many cases diverge from it. There is much that Tolkien intended to revise but only sketched out in notes, and some new texts surfaced after the publication of The Silmarillion. These books also make it clear just how unfinished the later parts of The Silmarillion really were: some parts were never rewritten after the early versions in Lost Tales.
The Silmarillion is a complex work exhibiting the influence of many sources. A major influence was the Finnish epic Kalevala, especially the tale of Kullervo. Influence from Greek mythology is also apparent. The island of Númenor, for example, recalls Atlantis. This, however, Tolkien later described in a letter to Christopher Bretherton as merely a "curious chance".
Greek mythology also colours the Valar, who borrow many attributes from the Olympian gods. The Valar, like the Olympians, live in the world, but on a high mountain, separated from mortals; But the correspondences are only approximate; the Valar also contain elements of Norse mythology. Several of the Valar have characteristics resembling various Æsir, the gods of Asgard. Thor, for example, physically the strongest of the gods, can be seen both in Oromë, who fights the monsters of Melkor, and in Tulkas, the physically strongest of the Valar. Manwë, the head of the Valar, exhibits some similarities to Odin, the "Allfather". Tolkien also said that he saw the Maia Olórin (Gandalf) as an "Odinic wanderer".
Influence of the Bible and traditional Christian narrative are seen in The Silmarillion in the conflict between Melkor and Eru Ilúvatar, a parallel of the polarity of Lucifer and God. Further, The Silmarillion tells of the creation and fall of the Elves, as Genesis tells of the creation and fall of Man. As with all of Tolkien's works, The Silmarillion allows room for later Christian history, and one draft even has Finrod, a character in The Silmarillion, speculating on the necessity of Eru's (God's) eventual Incarnation to save mankind.
Medieval Christian cosmology shows its influence especially in the account of the creation of the universe as the manifestation of a sort of song sung by God with which the angels harmonize until the fallen angel introduces discord. St. Augustine's writings on music, as well as the extensive medieval tradition of the divine harmony—more familiar to us today in the notion of the "music of the spheres"—served as bases for this telling of creation.
Celtic mythology show its influence in the exile of the Noldorin Elves, for example, that borrow elements from the story of Irish legends of the Tuatha Dé Danann. Welsh influence is seen in the Elvish language Sindarin, that Tolkien gave "a linguistic character very like (though not identical with) British-Welsh ... because it seems to fit the rather 'Celtic' type of legends and stories told of its speakers".
At the time of release, reviews of The Silmarillion were generally mixed to negative. The Silmarillion was criticized for being too serious, lacking the light-hearted moments that were found in The Lord of the Rings and especially The Hobbit. Time magazine lamented that there was "no single, unifying quest and, above all, no band of brothers for the reader to identify with". Other criticisms included difficult-to-read archaic language and many difficult and hard-to-remember names.
Robert M. Adams of The New York Review of Books called The Silmarillion "an empty and pompous bore", "not a literary event of any magnitude", and even claimed that the main reason for its "enormous sales" was the "Tolkien cult" created by the popularity of The Hobbit and The Lord of the Rings, predicting that more people would buy The Silmarillion than would ever read it. The School Library Journal called it "only a stillborn postscript" to Tolkien's earlier works. Peter Conrad of the New Statesman even went so far as to say that "Tolkien can't actually write".
Nonetheless, a few reviewers praised the scope of Tolkien's creation. The New York Times Book Review acknowledged that "what is finally most moving is … the eccentric heroism of Tolkien's attempt". TIME described The Silmarillion as "majestic, a work held so long and so powerfully in the writer's imagination that it overwhelms the reader". The Horn Book Magazine even lauded the "remarkable set of legends conceived with imaginative might and told in beautiful language". John Calvin Batchelor, reviewing the book for The Village Voice, lauded the book as a "difficult but incontestable masterwork of fantasy" and praised the character of Melkor, describing him as "a stunning bad guy" whose "chief weapon against goodness is his ability to corrupt men by offering them trappings for their vanity".
The Dutch progressive rock guitarist Jan Akkerman recorded a track called "The Silmarillion" on his 1977 album Meditation.
The British neo-progressive rock band Marillion were formed in Aylesbury, England, in 1979 as Silmarillion but shortened their name in 1981 to avoid any trademark conflict.
The Austrian black metal band Summoning formed in 1993 and have also released numerous concept albums based on this and other works by Tolkien.
The German power metal band Blind Guardian released Nightfall in Middle-Earth in 1998, a concept album based upon The Silmarillion. The album contains not only songs but also spoken passages narrating parts of the story.
The German Drum & Bass DJ Mathis Mootz released War of Sound in 2003, a clicks n' cuts side project as m2, which contains spoken word samples from an adaptation of The Silmarillion.
The Irish Celtic metal band Cruachan released a song called the "Fall of Gondolin" on their 2004 album Pagan.
The Australian indie band The Middle East incorporate many elements from The Silmarillion in their song "Beleriand" from their 2009 album The Recordings of the Middle East.
The Swedish a cappella group Riltons Vänner incorporates loose influences of The Silmarillion in the lyrics of their song "Folket från Eä" (English: "The people from Eä"), written by Peter Sydén, from their 2005 album Här är passion.
The Norwegian classical composer Martin Romberg has written three full-scale symphonic poems, Quendi (2008), Telperion et Laurelin (2014), and Fëanor (2017), inspired by passages from The Silmarillion. The works were premiered by orchestras in Southern France between 2009 and 2017.
^ Rayner Unwin (1978), in Amon Hen, bulletin of The Tolkien Society, no. 32 p. 13.
^ Fimi, Dimitra (August 2006). "'Mad' Elves and 'Elusive Beauty': Some Celtic Strands of Tolkien's Mythology". Folklore, 117(2), 156–170 . JSTOR 30035484.
^ Batchelor, John Calvin (10 October 1977). "Tolkien Again: Lord Foul and Friends Infest a Morbid but Moneyed Land". The Village Voice. Retrieved 13 February 2013.
^ "Martin Romberg at Orchestre régional Avignon-Provence". Orchestre régional Avignon-Provence.
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IL-17 Production by γδ+ T Cells Is Critical for Inducing Th17 Responses in the Female Genital Tract and Regulated by Estradiol and Microbiota
Varun C. Anipindi, Puja Bagri, Sara E. Dizzell, Rodrigo Jiménez-Saiz, Manel Jordana, Denis P. Snider, Martin R. Stämpfli and Charu Kaushic
ImmunoHorizons July 1, 2019, 3 (7) 317-330; DOI: https://doi.org/10.4049/immunohorizons.1900040
Varun C. Anipindi
*McMaster Immunology Research Centre, McMaster University, Hamilton, Ontario L8S 4K1, Canada; and
†Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario L8S 4K1, Canada
Puja Bagri
Sara E. Dizzell
ORCID record for Sara E. Dizzell
Rodrigo Jiménez-Saiz
ORCID record for Rodrigo Jiménez-Saiz
Manel Jordana
Denis P. Snider
ORCID record for Denis P. Snider
Martin R. Stämpfli
Charu Kaushic
ORCID record for Charu Kaushic
IL-17 can be produced by adaptive immune cells such as Th17 cells and by immune cells that produce IL-17 without prior priming. This latter category, which we will refer to as "innate," includes innate cells such as NK cells and innate lymphoid cells and innate-like T cell populations such as NKT cells and γδ+ T cells. Studies in mucosal tissues have shown that the induction of Th17 immunity is amplified by innate IL-17 produced within those tissues. However, the role of innate IL-17 and its effect on Th17 induction in the female genital tract (FGT) is largely unknown. In this study, we characterize the primary source of IL-17–secreting vaginal cells and show that innate IL-17 plays a critical role in priming adaptive Th17 responses in the FGT. Under homeostatic conditions, γδ+ T cells were the predominant source of innate IL-17 in the murine FGT, and this population was modulated by both the sex hormone estradiol and the presence of commensal microbiota. Compared with wild-type C57BL/6 mice, vaginal APCs isolated from IL-17A–deficient (IL-17A−/−) mice were severely impaired at priming Th17 responses in APC–T cell cocultures. Furthermore, the defect in Th17 induction in the absence of innate IL-17 was associated with impairment of IL-1β production by vaginal CD11c+ dendritic cells. Overall, our study describes a novel role for IL-17 in the FGT and further demonstrates the importance of factors in the vaginal microenvironment that can influence adaptive immune responses.
IL-17 belongs to a family of cytokines that consists of six related ligands (IL-17A to IL-17F) (1). IL-17 is primarily secreted by CD4+ T cells that differentiate into Th17 effector cells under the influence of cytokine combinations comprised of IL-6, IL-1β, TGF-β, IL-21, and IL-23 (2–5). The effector response of IL-17 involves the induction of inflammatory mediators such as TNF-α, IL-1, IL-8, G-CSF, and the recruitment of neutrophils for pathogen clearance (6–8). Th17 immunity and the production of IL-17 have been shown to play a significant role in the resolution of bacterial and fungal infections in mucosal tissues (9, 10), while demonstrating a pathological role in autoimmune and chronic inflammatory diseases (6, 11–13).
Within the female genital tract (FGT), a mucosal site exposed to diverse pathogens, IL-17 has been shown to play an important role during bacterial and fungal infections, including Neisseria gonorrheae, Candida albicans, and Chlamydia trachomatis (14–19). However, the role of IL-17 during genital viral infections has not been fully elucidated. We recently described a novel antiviral role for IL-17 in the FGT using a mouse model of genital herpes infection (20, 21). Genital herpes, predominantly caused by HSV type 2 (HSV-2), is one of the most prevalent sexually transmitted viral infections globally and affects more women than men (22). The primary protective response against HSV-2 involves the production of IFN-γ by CD4+ T cells in the FGT (23–25). However, we recently showed that enhanced protection against vaginal HSV-2 infection also coincided with increased levels of IL-17 production by CD4+ T cells (21). We found that mice treated with the female sex hormone estradiol (E2) were completely protected against genital HSV-2 challenge, and protection in these animals was related to increased Th17 immunity (21). Along with augmented Th17 responses, mice also demonstrated enhanced Th1 responses (21). Furthermore, in the absence of Th17 responses, vaccinated IL-17A–deficient mice (IL-17A−/−) had impaired IFN-γ+ CD4+ T cell responses as well as poor disease outcomes against HSV-2 challenge compared with wild-type (WT) C57BL/6 mice (20). Collectively, these studies suggest a significant role for Th17 cells in the immune response to viral infections in the FGT.
Interestingly, although Th17 cells are often associated with IL-17 production, other immune cells can also produce IL-17 without any prior priming. This production of IL-17, which we will refer to as innate IL-17, comes from sources such as innate cells (NK cells and innate lymphoid cells [ILCs]) and innate-like T cell populations (NKT cells and γδ+ T cells) (26). Although innate and innate-like cells represent a relatively small proportion of the responding lymphocytes to mucosal infections, they are more potent at producing IL-17 compared with Th17 cells (27). Specifically, innate-like γδ+ T cells are one of the earliest identified sources of IL-17 (28) and have been described as the major source of IL-17 under homeostatic conditions in the FGT (29). They develop early in the fetus and provide immunity prior to the generation of the adaptive immune system (30), and unlike traditional T lymphocytes, γδ+ T cells use their TCR directly as pattern recognition receptors to respond to pathogens (31, 32). γδ+ T cells are predominantly localized in mucosal tissues and rapidly activate upon exposure to pathogens and proinflammatory cytokines released by APCs (32). IL-1, IL-6, IL-18, IL-23, and TGF-β have all been shown to be important for IL-17 production by γδ+ T cells (28).
Production of innate IL-17 under homeostatic conditions has been implicated in the amplification of Th17 responses in mucosal tissues (33–37). In models of autoimmune encephalomyelitis (37) and colitis (33), IL-17+ γδ+ T cells promoted Th17 differentiation and amplified IL-17 production by Th17 cells. In our own studies, we have observed that vaginal cells can also constitutively produce IL-17 without any additional stimulation in vitro (21). This suggests that innate IL-17 may be an important component of the vaginal microenvironment and can potentially affect induction of Th17 responses. These findings prompted us to further determine the role of IL-17 in the FGT.
In the current study, we examined the importance of innate IL-17 during the induction of adaptive Th17 responses in the FGT. Similar to previous studies (20, 21), we found that in the absence of IL-17, mice were unable to initiate protective antiviral immunity and could not effectively clear HSV-2 virus in vivo. We then examined the ability of IL-17A−/− mice to induce an adaptive Th17 response in vitro and found that vaginal dendritic cells (DCs) from IL-17A−/− mice were significantly impaired at priming Th17 responses, and this was related to attenuated IL-1β production. We further characterized the innate IL-17–secreting cells and found that under homeostatic conditions, γδ+ T cells were the predominant source of IL-17 in the FGT. Finally, we demonstrated that IL-17 production by γδ+ T cells was modulated by the presence of female sex hormone E2 and commensal microflora. These data suggest that production of IL-17 in the vaginal tract under homeostatic conditions is critical for inducing Th17 responses, thereby amplifying the overall IL-17 response in the FGT.
C57BL/6 mice were obtained from Charles River Laboratories (Saint-Constant, QC, Canada). IL-17A−/− mice were kindly provided by Dr. M. Stämpfli (acquired with a Material Transfer Agreement from Dr. Y. Iwakura) (University of Tokyo, Japan) (38, 39), and E2 receptor α knockout (KO) mice (ERKO) were kindly provided by Dr. P. Chambon (University de Strasbourg, France) (40) and bred in the Central Animal Facility (McMaster University, Hamilton, Canada). Germ-free (GF) mice were bred in-house and purchased from the Farncombe Family Axenic-Gnotobiotic Facility (McMaster University). OVA receptor transgenic (Tg) mice (OT-II Tg) were purchased from The Jackson Laboratory (Bar Harbor, ME) (41). All mice were maintained under specific pathogen-free and standard temperature-controlled conditions, which followed a 12-h light/dark cycle. Routine quality assurance was done by serology and PCR to ensure mice remained specific pathogen-free and included testing dirty bedding sentinels, direct resident animals, and exhaust air duct samples of racks. All animal studies were approved by, and in compliance with, the Animal Research Ethics Board at McMaster University.
Hormone treatment
For hormone experiments, mice were ovariectomized (OVX) to deplete endogenous hormones according to previously published protocols (42, 43). Briefly, mice were administrated an injectable anesthetic preparation of ketamine and xylazine i.p. Ovaries were removed by making two bilateral incisions, followed by small incisions through the peritoneal wall, and excised through the incisions. Incisions were closed using surgical clips, and mice recovered for 14 d before the start of experiments. Two weeks later, OVX mice were anesthetized with injectable anesthetic (ketamine and xylazine) and implanted s.c. with 21-d release 17β-E2 (476 ng/mouse per day) or placebo (mock) pellets (Innovative Research of America, Sarasota, FL), according to previously published protocols (43). The level of serum E2 resulting from the pellets has previously been shown to correspond to that measured during the estrus cycle (44).
OVX mice were immunized intranasally with an attenuated strain of HSV: thymidine kinase–deficient (TK-) HSV-2. Briefly, mice were anesthetized using isoflurane and then inoculated with 5 μl of TK- HSV-2 (102 PFU/mouse) into each nare with a micropipette. Six weeks later, mice were challenged intravaginally with WT HSV-2 (5 × 103 PFU/mouse). Vaginal washes were collected for 6 d postchallenge by pipetting 30 μl of PBS into the vagina five to six times and repeated to collect a total volume of 60 μl.
Genital pathology scoring
Genital pathology was monitored daily based on a five-point scale, as described previously (20, 21): no infection (0), slight redness of external vagina (1), swelling and redness of vagina (2), severe swelling and redness of vagina and surrounding tissues (3), genital ulceration with severe redness and hair loss (4), and severe ulceration extending to surrounding tissues, ruffled hair, hunched back, and lethargy. Animals were sacrificed before they reached stage 5.
Viral titration
Viral shedding in vaginal washes was determined by conducting viral plaque assays on Vero cell (American Type Culture Collection, Manassas, VA) monolayers. Vero cells were grown in α-MEM (Life Technologies, Burlington, ON, Canada) supplemented with 5% FBS (Life Technologies), 1% penicillin-streptomycin (Invitrogen, Burlington, ON, Canada), l-glutamate (BioShop Canada, Burlington, ON, Canada), and 1% HEPES (Invitrogen). Cells were grown to confluency in 12-well plates. Vaginal washes were diluted in α-MEM and then added to monolayers. Infected monolayers were incubated at 37°C for 2 h. Infected monolayers were then overlaid with α-MEM, and infection was allowed to occur for 48 h at 37°C. Cells were then fixed and stained with crystal violet, and viral plaques were enumerated under a microscope. The number of PFU/milliliter was calculated by taking a plaque count for every sample and accounting for the dilution factor.
Tissue isolation and cocultures
Vaginal tissue was enzymatically digested in 15 ml of RPMI 1640 containing 0.00157 g/ml collagenase A (Roche Life Science) at 37°C on a stir plate for 2 h and filtered through a 40-μm filter to obtain a total tissue cell (TC) suspension (45). OT-II Tg spleens were mechanically disrupted, and ammonium-chloride-potassium lysing buffer (Sigma-Aldrich, St. Louis, MO) was used to lyse blood cells. Mononuclear cells were counted, and CD4+ T cells were magnetically sorted using CD4 L3TE microbeads (Miltenyi Biotec, Auburn, CA) based on manufacturer protocols. Sorted CD4+ T cells were stained with 50 μM CFSE (Sigma-Aldrich) according to published protocols (46). Vaginal TC (5 × 105 cells/ml) were incubated in a 96-well plate pulsed with 5 μg/well OVA peptide (323–339) (Biomer Technology, Pleasanton, CA) with sequence ISQAVHAAHAEINEAGR for 16–18 h. CFSE-stained OT-II Tg CD4+ T cells were added at a 1:1 ratio and cocultured for 3.5 d at 37°C in complete RPMI media (47), based on previously standardized protocols (48). During certain experiments, 100 ng/ml rIL-1β or 250 ng/ml rIL-17 (R&D systems, Minneapolis, MN) was added during the peptide-pulse stage or on the first day of coculture.
For IL-17A or IL-1β intracellular staining, freshly isolated vaginal TCs or 2-d-old cocultures were treated with 2 μl/ml cell stimulation mixture plus 500× protein transport inhibitors: a mixture of PMA, ionomycin, brefeldin A, and monensin (eBioscience, San Diego, CA) for 16 h at 37°C. Cells were stained with allophycocyanin-ef780 viability dye (eBioscience) for 30 min to exclude dead cells and subsequently stained with the following surface Abs: CD3 BV785 (BioLegend, San Diego, CA), CD4 BV421 (BioLegend), TCR γδ PE (eBioscience), NKp46 PE-cf594 (eBioscience), CD11c PE-Cy7 (BD Biosciences, Mississauga, ON, Canada), or CD11b PE-cf594 (BD Biosciences). Cells were then permeabilized and fixed with BD Pharmingen Transcription Factor Buffer Set (BD Biosciences) according to manufacturer protocols and stained with the following intracellular Abs: IL-17A allophycocyanin (eBioscience), IL-1β PE (R&D Systems), and pro–IL-1β PerCP-ef710 (eBioscience) or control rat IgG1 allophycocyanin (eBioscience), rat IgG1 PE (R&D Systems), and rat IgG1 PerCP-ef710 (eBioscience). Data were acquired on a BD LSR II Flow Cytometer System (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR).
Cytokine analysis
IL-17 levels in coculture supernatants were measured using the Duoset ELISA kit (R&D Systems), according to manufacturer protocols, and plates were analyzed on a Sector Imager 2400 (Meso Scale Discovery, Rockville, MD).
Statistical analysis and graphical representations were performed using GraphPad Prism 6.0d (GraphPad Software, San Diego, CA). Data are expressed as mean ± SEM, typically derived from n = 3 replicates. Significance was calculated by comparing means using one-way or two-way ANOVA or t tests, as indicated in the figure legends. Statistical significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
All animal use was conducted in accordance to protocols approved by the McMaster University Animal Research Ethics Board as per Animal Utilization Protocol no. 14-09-40 in accordance with Canadian Council of Animal Care guidelines.
Innate IL-17 in the FGT plays an important role in inducing Th17 responses
Although studies in other mucosal sites, such as the gut, have shown that innate and innate-like sources of IL-17 can help amplify the overall IL-17 response by enhancing Th17 responses (33–37), a similar role for IL-17 in the FGT has not been yet reported. Therefore, we wanted to examine the effect of innate IL-17 on Th17 cell priming by vaginal DCs.
First, to confirm the in vivo significance of vaginal IL-17 in the context of a viral infection, we used a mouse model of HSV-2 immunization and challenge to examine the survival of mice in the absence of IL-17, similar to our previous studies (20, 21). IL-17A−/− mice and WT C57BL/6 controls were OVX, immunized intranasally with an attenuated strain of TK- HSV-2, and then challenged intravaginally with WT HSV-2. Survival and genital pathology were monitored daily, and vaginal washes were collected to measure viral shedding and determine disease severity. Consistent with our previous observations (20, 21), following genital HSV-2 challenge, IL-17A−/− mice had poor disease outcomes and demonstrated greater mortality (Fig. 1). Only 17% of IL-17A−/− mice survived the viral challenge, compared with 83% of WT controls by day 12 (Fig. 1A). All mice that reached end point developed genital pathology (IL-17A−/−: 5/6 mice; WT: 1/6 mice) (Fig. 1B). Similarly, viral shedding was also significantly higher in IL-17A−/− animals (p = 0.0281) (Fig. 1C), suggesting that IL-17 plays an important role in modulating antiviral responses in the FGT and confirming our previous findings (20, 21).
IL-17A−/− mice are more susceptible to intravaginal HSV-2 challenge following intranasal immunization.
OVX WT (C57BL/6) and IL-17A−/− mice (n = 6 per group) were immunized intranasally with TK- HSV-2 (102 PFU/mouse) and 6 wk later challenged intravaginally with WT HSV-2 (5 × 103 PFU/mouse). Survival and genital pathology were monitored, and vaginal washes were collected daily for 6 d postchallenge. (A) Significance in difference in survival was calculated using the log rank (Mantel-Cox) test (*p < 0.05). (B) Genital pathology scores were recorded on a scale of 0 to 5. Data points superimposed on the x-axes of (B) indicate mice without genital pathology, and the percentages represent maximum numbers of mice that demonstrated pathology. Each symbol represents a single animal. (C) HSV-2 shedding was calculated using a Vero cell-based plaque assay, and data represent the viral loads (means ± SEMs) over 6 d. Data were analyzed using an unpaired t test with 95% confidence interval (*p < 0.05). Data shown are representative of three independent experiments with similar results.
Next, we wanted to determine the effect of innate IL-17 production on the induction of Ag-specific Th17 responses in the FGT. We used a previously well-described, in vitro chicken OVA peptide coculture model with OVA-specific OT-II Tg CD4+ T cells (41). Vaginal TC, including APCs, were isolated from IL-17A−/− mice and WT C57BL/6 controls, cultured in medium alone or pulsed with OVA peptide, and cocultured with CFSE-labeled OT-II Tg CD4+ T cells (TC+CD4) for 3.5 d, as previously described (21). Using this model, we measured IL-17 levels in TC-alone cultures and found that WT mice produced innate IL-17 (100 ± 43 pg/ml), whereas IL-17 was below detection in TC cultures from IL-17A−/− mice (p = 0.003) (Fig. 2A). To determine the role of innate IL-17 on Th17 differentiation, IL-17 levels were measured in TC+CD4 coculture supernatants. Cultures containing TC from IL-17A−/− mice contained 30-fold lower IL-17 compared with those with TC from WT mice (p = 0.001) (Fig. 2A). In addition, we conducted intracellular cytokine staining on day 2 of the cocultures and found that a lower proportion of proliferating CFSE-stained OT-II Tg CD4+ T cells expressed IL-17 in IL-17A−/− TC+CD4 cocultures (39%) compared with WT controls (55%) (Fig. 2B). These observations provide further evidence that the absence of innate IL-17 results in diminished Th17 responses. The small population of CFSE− cells within WT TC+CD4 cocultures (3.87%) likely represented the innate IL-17 population in the FGT that was entirely absent in IL-17A−/− cocultures (Fig. 2B). The innate IL-17 population had a ∼30-fold higher median fluorescence intensity (MFI) compared with the CFSE+ Th17 cells (p = 0.040), indicating that the CFSE− population of IL-17–secreting cells was a more potent source of IL-17 than Th17 cells on a per cell basis (Fig. 2C).
IL-17 produced by innate sources in the FGT is critical for potentiating Th17 responses primed by vaginal APCs.
Vaginal cells from hormone cycle–matched WT (C57BL/6) or IL-17A−/− mice (n = 5) were pooled, pulsed with chicken OVA peptide, and cultured alone (TC) or cocultured at a 1:1 ratio with CFSE-stained OT-II Tg CD4+ T cells (TC+CD4) for 3.5 d. (A) IL-17 levels in culture supernatants were measured by ELISA. (B) On day 2 of coculture, a cell stimulation mixture containing Golgi inhibitors and PMA plus ionomycin was added, and 18 h later, cocultures were stained with Abs against CD3, CD4, and IL-17 and analyzed by flow cytometry to identify IL-17+ cells and measure MFI. Upper box indicates CFSE−, IL-17–producing cells in culture. The lower box indicates CFSE+, IL-17–producing CD4+ T cells, with a lower cut-off that excludes 95% of isotype control CSFE+ cells. (C) The difference in MFI was compared between innate and Th17 sources of IL-17 (n = 3 independent experiments). Data were analyzed using an unpaired t test with 95% confidence interval (*p < 0.05). (D) Similar cocultures were conducted, with 250 ng/ml of rIL-17 added during the peptide-pulse stage (TCIL-17p+CD4) and washed away before coculture or added on day 1 of coculture (TC+CD4+IL-17) and remained present throughout the duration of the experiment as indicated on the x-axis. IL-17 levels in culture supernatants were measured by ELISA. Data in (A) and (D) are mean ± SEM of three individual wells per coculture condition. Data are representative of three independent experiments with similar results. Significance was calculated by two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance).
Given the attenuated priming of Th17 responses demonstrated by IL-17A−/− vaginal TCs (Fig. 2A), we wanted to examine whether exogenous addition of rIL-17 could rescue the ability of DCs within the IL-17A−/− vaginal TCs to induce Th17 responses. Vaginal cells from IL-17A−/− mice and WT mice were isolated, pulsed with OVA peptide, and cocultured with OT-II Tg CD4+ T cells. To determine whether IL-17 is only required transiently as a pretreatment, 250 pg/ml rIL-17 (a level consistent with that observed in cultures of vaginal cells from WT controls alone [Fig. 2A]) was added during the OVA peptide pulse and removed 12 h later by washing, before coculturing with OT-II Tg CD4+ T cells. Alternatively, to examine whether prolonged exposure of IL-17 is required during the process of T cell differentiation, rIL-17 was added at day 1 of the coculture and remained in culture for the duration of the experiment. In both conditions, rIL-17 was unable to significantly influence Th17 responses in IL-17A−/− cocultures (Fig. 2D), suggesting that short-term restoration of IL-17 may not be sufficient to restore the ability of IL-17A−/− vaginal APCs to prime Th17 responses. Taken together, these results affirm the importance of IL-17 in the antiviral response in the FGT and show that innate IL-17 within the FGT is important for the induction of Th17 responses.
Innate IL-17 induces vaginal DCs to prime Th17 responses via an IL-1β–dependent pathway
Various cytokines have been implicated for IL-17 production (28). In our own studies, we showed that within the FGT, adaptive IL-17 production by Th17 cells was primed by CD11c+ DCs via an IL-1–dependent pathway (21). Given the attenuated Th17 responses observed in cocultures containing vaginal APCs from IL-17A−/− mice (Fig. 2), we wanted to examine whether innate IL-17 plays a role in programming vaginal APCs to produce IL-1.
Vaginal TCs from IL-17A−/− mice and WT controls were isolated and cultured overnight (12 h) prior to staining with Abs against CD11c, CD11b, and IL-1β. Dead cells were excluded using a fixable viability dye, CD11c+ DCs were gated (Fig. 3A), and intracellular IL-1β expression was compared by flow cytometry. We found that IL-17A−/− mice contained a significantly lower proportion of IL-1β–producing CD11c+ DCs in the FGT compared with WT mice (p = 0.04) (Fig. 3B, 3C). In addition, CD11c+ DCs from IL-17A−/− mice also contained reduced amounts of IL-1β per cell compared with WT controls, based on MFI (Fig. 3D), although not statistically significant (p = 0.400).
Vaginal CD11c+ DCs from IL-17A−/− mice are impaired in potentiating Th17 responses because of diminished IL-1β production.
Vaginal cells from hormone cycle–matched WT or IL-17A−/− mice (n = 5) were pooled, cultured overnight without any additional stimulation, stained with Abs against CD11c, CD11b, and IL-1β, and analyzed by flow cytometry to identify IL-1β+ cells, with dead cells excluded. (A) CD11c+ DCs were gated as shown, and (B) IL-1β expression by vaginal CD11c+ CD11b+ DCs from WT and IL-17A−/− mice was compared with isotype control. (C) The differences in percentage and (D) MFI of IL-1β+ DCs were compared between vaginal cells from WT and IL-17A−/− mice (n = 3 independent experiments). Data were analyzed using an unpaired t test with 95% confidence interval (*p < 0.05). (E) Vaginal cells from hormone cycle–matched WT or IL-17A−/− mice (n = 5) were pooled, pulsed with chicken OVA peptide, and cultured alone (TC) or cocultured at a 1:1 ratio with OT-II Tg CD4+ T cells (TC+CD4) for 3.5 d. Alternately, 100 ng/ml of rIL-1β was added on day 1 of coculture (TC+CD4+IL-1β). IL-17 levels in culture supernatants were measured by ELISA. Data are mean ± SEM of three individual wells per coculture condition. Data are representative of three independent experiments with similar results. Significance was calculated by two-way ANOVA (*p < 0.05, ***p < 0.001, ****p < 0.0001).
Given this observation, we wanted to examine whether the addition of exogenous IL-1β would restore Th17 responses in IL-17A−/− cocultures. Addition of rIL-1β to IL-17A−/− cocultures on day 1 (TC+CD4+IL-1β) was able to significantly (p = 0.0005) enhance Th17 responses almost 10-fold compared with cultures without IL-1β (Fig. 3E). However, IL-17 levels were still significantly lower than those seen in cultures with WT vaginal TCs (Fig. 3E).
Overall, these results showcase an inherent deficit in the ability of vaginal DCs from IL-17A−/− mice to produce IL-1β, thereby suggesting that the priming of Th17 responses by innate IL-17 is dependent on IL-1β. Even with the addition of exogenous IL-1β, there was only a partial rescue of Th17 responses primed by IL-17A−/− vaginal DCs. Together, this suggests that innate IL-17 is important for programming vaginal DCs to produce IL-1β, which in turn is critical for priming vaginal Th17 responses in the FGT.
γδ+ T cells are the predominant source of innate IL-17 in the FGT
Having established an important role for innate IL-17 in the priming of Th17 responses in the FGT, we next wanted to characterize the primary sources of IL-17 production. IL-17 in mucosal tissues can be produced by a variety of innate lymphocytes (NK cells and ILCs) (26), innate-like lymphocytes (NKT and γδ+ T cells) (26), and adaptive sources (Th17 cells) (49), and so we focused on examining these lymphocyte populations.
Vaginal cells isolated from C57BL/6 mice were stained with a panel of Abs against CD3, CD4, TCR γδ, IL-17, and NKp46 to identify these lymphocyte populations (29, 50). Dead cells were excluded using a fixable viability dye, and single cells were gated to identify total IL-17+ populations using flow cytometry (Fig. 4A). We found that IL-17+ cells did not express NKp46 (Fig. 4A) and could instead be divided into two major populations based on their CD3 and CD4 expression (CD3+ CD4+ and CD3+ CD4−) (Fig. 4B). These were further separated based on TCR γδ expression (Fig. 4B), and we observed that γδ+ T cells (CD3+ CD4− γδ+) were the primary source of IL-17 (Fig. 4C, 4D). The γδ+ T cell population accounted for 75% of total IL-17–producing cells, which was significantly higher than both CD4+ T cells (21%) and other sources (4%) of IL-17 (Fig. 4C, 4D).
IL-17 in the FGT is predominantly produced by γδ+ T cells.
Vaginal cells from hormone cycle–matched WT (C57BL/6) or IL-17A−/− mice (n = 5–7) were isolated, pooled, and stimulated with a cell stimulation mixture containing Golgi inhibitors and PMA plus ionomycin for 18 h. Cells were stained with Abs against CD3, CD4, NKp46, TCR γδ, and IL-17 and analyzed by flow cytometry to identify IL-17+ cells. (A) Dead cells were excluded, and total IL-17+ cells were gated. (B) Subsets of IL-17+ cells were further gated based on CD3 and CD4 expression, and each subset was examined for TCR γδ expression. (C) The distribution of cell subsets among all IL-17–producing cells were examined and (D) quantified in five independent experiments. Data are mean ± SEM, and significance was calculated by one-way ANOVA (****p < 0.0001). (E) The difference in percentage of γδ+ cells was compared between WT and IL-17A−/− mice from four independent experiments. Data were analyzed using an unpaired t test with 95% confidence interval. Data are representative of four independent experiments with similar results. ns, no significance.
We also examined TCR γδ expression by vaginal cells from IL-17A−/− mice and found frequencies of γδ+ cells comparable to those in WT animals (Fig. 4E) (not significant; p = 0.243), suggesting no inherent defect in the development of the γδ+ population in the absence of IL-17. Similar to previous reports (29), these findings confirm that innate-like γδ+ T cells represent the primary source of IL-17 in the FGT under homeostatic conditions.
E2 enhances IL-17 production by γδ+ T cells in the FGT
The FGT is a complex tissue, regulated by a variety of factors within the local microenvironment. One major component that influences immune cell populations and responses in the FGT is the presence of female sex hormones. Specifically, we have shown that the female sex hormone E2 is protective against viral infections in the FGT and can enhance Th17 responses primed by vaginal CD11c+ DCs (21). Therefore, we wanted to examine whether E2 can also influence innate IL-17+ populations in the FGT.
To determine the effect of E2 on the primary IL-17–secreting cell population (γδ+ T cells), C57BL/6 mice were OVX to remove endogenous sources of reproductive hormones and implanted with prolonged-release E2 or placebo (mock) pellets. Two weeks later, vaginal cells were isolated and stained for flow cytometry. We found that E2 treatment consistently and significantly (p = 0.05) induced greater proportions of IL-17+ cells in the FGT compared with mock-treated or ERKO controls (Fig. 5A, 5B). Furthermore, IL-17+ cells from E2-treated mice produced significantly greater IL-17 per cell (p = 0.007) compared with controls, based on MFI levels (Fig. 5C). When we characterized the overall IL-17+ population based on CD3 and CD4 expression, and further separated these populations based on TCR γδ expression as done previously (Fig. 4), we found that the predominant source of IL-17 in E2-treated animals was the γδ+ T cell population (Fig. 5D–F) described earlier (Fig. 4). However, this population was largely absent or diminished in mock-treated mice (E2: 64 ± 12%; mock: 16 ± 6%; p = 0.02) (Fig. 5F).
E2 induces IL-17–producing γδ+ T cells in the FGT.
Vaginal cells from OVX WT (C57BL/6) mice treated with E2 or placebo (mock) pellets (n = 5) were isolated, pooled, and stimulated with a cell stimulation mixture containing Golgi inhibitors and PMA plus ionomycin for 18 h. (A) Cells were stained with Abs against CD3, CD4, and IL-17 and analyzed by flow cytometry. ERKO mice were used as additional controls. Dead cells were excluded, and total IL-17+ cells were gated. (B) The differences in percentage and (C) MFI of IL-17+ cells were compared between E2 and mock mice from three independent experiments. Data were analyzed using the unpaired t test with 95% confidence interval (*p < 0.05, **p < 0.01). (D) Subsets of IL-17+ cells were further identified based on CD3 and CD4 expression. (E) The distributions of the cell subsets among all IL-17–producing cells comparing E2-treated and mock-treated mice. (F) The difference in percentage of IL-17–producing γδ+ cells was compared between E2 and mock mice from three independent experiments. Data are mean ± SEM, and significance was calculated using an unpaired t test with 95% confidence interval (*p < 0.05). Data are representative of three independent experiments with similar results.
Taken together, these findings demonstrate that factors in the microenvironment of the FGT, such as the presence of E2, can influence innate levels of IL-17. Specifically, E2 plays a critical role in upregulating IL-17 production by γδ+ T cells.
Commensal microbiota enhances IL-17 production by γδ+ T cells in the FGT
Another factor shown to influence IL-17 production in mucosal tissue is the presence of the commensal microflora. In the gut, the local microbiota has been reported to drive IL-17 production by both γδ+ T cells (51, 52) and Th17 cells (53). Similarly, the vaginal microflora has also been shown to regulate immune responses in the FGT (54). As a result, we investigated whether the vaginal microbiota could also influence innate IL-17 in the FGT.
To determine the effect of the microbiota on the primary IL-17–secreting cell population (γδ+ T cells), vaginal cells from hormone cycle–matched C57BL/6 GF mice and WT mice were isolated, and IL-17 production was compared by flow cytometry. We consistently found that GF mice had significantly lower proportions of total IL-17+ cells compared with WT controls (p = 0.028) (Fig. 6A, 6B) as well as lower IL-17 produced per cell (p = 0.002) (Fig. 6C). Subsequently, a distribution of IL-17–producing cell subtypes based on CD3 and CD4 expression (Fig. 6D), followed by TCR γδ expression (Fig. 6E), showed that IL-17+ γδ+ T cells were significantly decreased in GF mice (GF: 26 ± 4%; WT: 54 ± 8%; p = 0.02) (Fig. 6F). Overall, these results demonstrate that presence of microbiota enhances IL-17 production by γδ+ T cells in the FGT.
Microbiota induces IL-17–producing γδ+ T cells in the FGT.
Vaginal cells from hormone cycle–matched WT (C57BL/6) or GF mice (n = 5) were isolated, pooled, and stimulated with a cell stimulation mixture containing Golgi inhibitors and PMA plus ionomycin for 18 h. (A) Cells were stained with Abs against CD3, CD4, and IL-17 and analyzed by flow cytometry. Dead cells were excluded, and total IL-17+ cells were gated. (B) The differences in percentage and (C) MFI of IL-17+ cells were compared between GF and WT mice from seven independent experiments. Data were analyzed using the unpaired t test with 95% confidence interval, with the ROUT method used to identify outliers (*p < 0.05, **p < 0.01). (D) Subsets of IL-17+ cells were further identified based on CD3 and CD4 expression. (E) The distributions of the cell subsets among all IL-17–producing cells comparing WT and GF mice. (F) The difference in percentage of IL-17–producing γδ+ cells was compared between WT and GF mice from four independent experiments. Data are mean ± SEM, and significance was calculated using an unpaired t test with 95% confidence interval (*p < 0.05). Data are representative of four independent experiments with similar results.
Although IL-17 is commonly referred to as a CD4+ T cell–secreted cytokine, the majority of IL-17 released constitutively under homeostatic conditions is produced by CD4− innate and innate-like immune cells. The presence of innate IL-17 in mucosal tissues has been shown to be important for potentiating Th17 immunity and thereby playing an important role in protecting against pathogen exposure and disease (33–37). In the FGT, Th17 responses have shown to be protective against bacterial, fungal, and viral infections (14–19). Recent work from our laboratory has focused on the role of Th17 responses during genital HSV-2 infection (20, 21). We have shown that vaginal DCs are critical for priming Th17 responses, which leads to enhanced Th1 responses (21), and that IL-17 plays a critical role in mediating Th1 responses during in vivo HSV-2 challenge (20, 21). However, the significance of IL-17 produced by innate or innate-like sources in the FGT and its influence on Th17 immunity is less understood. In the current study, we investigated the role of innate IL-17 in the induction of Th17 responses in the FGT and found that innate IL-17 was important for inducing vaginal Th17 responses. This was evident, as vaginal APCs from IL-17A−/− mice were severely impaired at priming Th17 responses in APC–T cell cocultures (Fig. 2). In addition, vaginal DCs from these mice also produced significantly lower levels of IL-1β (Fig. 3), which is critical for priming Th17 responses in the FGT (21). We further characterized the IL-17–secreting cells in the FGT and discovered that the predominant source was innate-like γδ+ T cells (Fig. 4). Furthermore, IL-17 production by γδ+ T cells was shown to be influenced by factors such as sex hormones (E2) and the microbiota (Figs. 5, 6). To the best of our knowledge, this is the first study demonstrating that innate IL-17 plays a critical role in potentiating Th17 immunity in the FGT and examining the factors that regulate it.
Numerous studies have shown that innate and innate-like lymphocytes can influence the differentiation and function of adaptive lymphocytes (55). We previously observed that vaginal cells can produce modest amounts of IL-17 in culture without any stimulation (21), and we wanted to further examine the influence of this innate IL-17 on the priming of Th17 responses by vaginal DCs. We consistently found that vaginal cells from IL-17A−/− mice were impaired at priming Th17 responses in vitro (Fig. 2A, 2D), suggesting that the presence of innate IL-17 can influence adaptive immunity primed by vaginal APCs. Interestingly, rIL-17 (250 pg/ml) did not restore the functional ability of the DCs in priming these responses (Fig. 2D). This is consistent with the observations made in a study by Kumar et al. (56) that provided evidence demonstrating that Th17 differentiation is not directly dependent on IL-17RA signaling and thereby suggests that IL-17 affects Th17 differentiation in an indirect manner. However, other in vivo studies suggest that the absence of IL-17 may lead to inherent defects in DC-mediated priming of CD4+ T cell responses (57). Bai et al. (57) demonstrated, using a lung C. muridarum infection model, that neutralization of IL-17 significantly impaired DC function, including lower levels of IL-12 production and lower expression of MHC class II and CD40. Furthermore, the mice produced higher levels of IL-10 and IL-4 and skewed the immune responses toward a Th2 phenotype instead of the typical Th1 response seen in controls (57). Likewise, we have previously shown that in the absence of IL-17 (IL-17A−/− mice), there was a 7-fold lower Th1 response in the FGT compared with WT mice (20, 21). Our current findings further expand on the idea that under homeostatic conditions, IL-17 plays a critical role in priming CD4+ T cell responses in the FGT.
The production of IL-17 has been shown to depend on different cytokines, including IL-1, IL-6, IL-21, IL-23, and TGF-β (2–5, 28). However, although γδ+ T cells from IL-6 KO and TGF-β KO mice produced comparable or attenuated levels of IL-17, respectively, those from IL-1R KO mice failed to secrete any IL-17 in response to IL-23 and TLR agonists (37, 58). This suggests that IL-1 may play a critical role in the production of IL-17 by γδ+ T cells. Likewise, we previously showed that IL-1β, but not IL-6, production by vaginal CD11c+ DCs is critical for priming Th17 responses in the FGT (21). Therefore, we wanted to examine whether the attenuated Th17 responses seen in IL-17A−/− cocultures was due to a defect in the ability of IL-17A−/− mice to produce IL-1β. We found that vaginal DCs from IL-17A−/− mice produced lower amounts of IL-1β (Fig. 3B, 3C) compared with WT DCs. These observations are directly consistent with prior literature indicating that IL-17 can induce IL-1β production in mucosal APCs (59, 60). Nakae et al. (39) observed lower IL-1β and impaired CD4+ T cell responses by Langerhans cells from IL-17A−/− mice. Furthermore, although the addition of rIL-1β increased Th17 differentiation in IL-17A−/− cocultures (Fig. 3E), these levels were still significantly lower than those observed in WT cultures. Given that there are numerous stimulatory and inhibitory factors involved in priming Th17 responses (61), we suggest that there may be other intrinsic defects in IL-17A−/− DCs related to signal transduction, activation markers (56), or cytokine processing (39) that cannot be restored by the transient addition of rIL-1β. Overall, our findings demonstrate that IL-17 in the FGT microenvironment may have an important effect on the function of vaginal DCs and their ability to prime adaptive Th17 responses via an IL-1β–dependent pathway.
Although innate IL-17 can be produced by a mixed population of lymphocytes under homeostatic conditions, consistent with a previous report by Kim et al. (29), we found that γδ+ cells are the primary source of innate IL-17 in the FGT (Fig. 4D). Additionally, we looked at γδ+ cells in the FGT of IL-17A−/− mice to determine if there was a defect in the γδ+ T cell population in the absence of IL-17 and found no significant difference as compared with WT mice (Fig. 4E). We also identified other sources of innate IL-17– producing cells (62). Although the CD3+ CD4+ IL-17+ cells are typically identified as Th17 cells, subpopulations in this subset can also represent innate cell types such as fetal lymphoid tissue inducer cells or NK cell receptor negative type 3 ILCs (50). Because of the lack of CD4 expression in CD3+ CD4− γδ − IL-17+ cells, these may represent NK cell receptor negative type 3 ILCs. However, given the previous observations by Serafini et al. (63) showing that GATA3 is important for type 3 ILCs, further studies with GATA3 KO mice may be required to conclusively identify these cells in the FGT. Irrespective of these other sources of IL-17, our findings demonstrate that γδ+ T cells are the primary source of innate IL-17 in the FGT.
Having identified the primary source of IL-17–producing cells critical for inducing adaptive Th17 responses, we examined how two important factors in the local FGT microenvironment, sex hormones, and the microbiota can influence the presence and function of these cells. We have previously shown that E2 can enhance Th1 and Th17 responses in the FGT (20, 21). However, the effect of E2 on levels of innate IL-17 has not been reported. We found that E2-treated mice contained a higher proportion of overall IL-17+ cells in the FGT compared with mock or ERKO control animals (Fig. 5A–C). Also, the primary source of this IL-17 in the FGT, γδ+ T cells, was almost completely absent in E2-depleted mice (Fig. 5D–F). These results suggest that E2 is involved in modulating and enhancing the production of innate IL-17.
There is also cross-talk between the microbiota and hormones present in the FGT, and E2 has been shown to be important for a healthy vaginal microflora (64, 65). Interestingly, the gut microbiota has been linked to IL-1β–mediated Th17 responses primed by mucosal DCs (66), and the microbiome has also been linked to IL-17 production by innate and innate-like lymphocytes in mucosal tissues (36, 67); however, the influence of the vaginal microbiota on IL-17 production is largely unknown. We examined whether the microbiota is important for IL-17 production in the FGT by using GF mice devoid of commensal bacteria, and additionally, we used stage-matched animals to ensure there was limited influence of hormones on the immune responses measured. We observed a significant reduction in the overall proportion of IL-17+ cells in the FGT of GF mice compared with WT controls (Fig. 6A–C), and there was also a marked decrease in the proportion of γδ+ T cells in these mice (Fig. 6E, 6F). These findings are in contrast to a previous report by Kim et al. (29) in which they concluded that the vaginal microflora does not affect IL-17 production by genital γδ+ T cells. However, they used antibiotics via drinking water to deplete commensal bacteria, whereas we used GF mice that were completely devoid of microflora. The effects of the temporary depletion compared with complete absence of bacteria during development of mucosal immunity may explain the discrepancy between our findings. Together, our findings in regard to the effects of E2 and microbiota demonstrate the degree to which the microenvironment can influence the induction of immune responses and the development, self-renewal, or maintenance of IL-17–producing γδ+ T cells in the FGT.
In summary, our study expands on previous work regarding the importance of antiviral Th17 immunity in the FGT (20, 21) and provides insight regarding a novel role for IL-17 in the vaginal microenvironment. We have shown that innate IL-17 is primarily secreted by γδ+ T cells in the FGT and is modulated by both E2 and the microbiota. This IL-17–rich environment conditions vaginal DCs to prime potent Th17 responses, likely via an IL-1β–dependent pathway. To our knowledge, this is the first report to show the importance of IL-17 produced by γδ+ T cells in the induction of Th17 immunity in the FGT as well as demonstrate an association between E2, microbiota, and IL-17–producing γδ+ T cells. Our findings showcase the complexity of the vaginal microenvironment and highlight the importance of understanding how endogenous factors (IL-17, hormones, microbiota) can regulate the priming of adaptive immune responses by vaginal DCs and thereby influence overall immunity in the FGT.
We thank Kristen Mueller for providing technical assistance.
This work was supported by operating grants from the Canadian Institutes of Health Research (to C.K.) (Funding Reference Number 93615) and an Applied HIV Research Chair Award (to C.K.) from the Ontario HIV Treatment Network (Applied HIV Research Chair 779). V.C.A. and P.B. were supported by Ontario Graduate Scholarships.
E2 receptor α knockout
HSV type 2
IL-17A−/−
IL-17A–deficient
innate lymphoid cell
median fluorescence intensity
OT-II Tg
OVA receptor transgenic mice
ovariectomized
TK-
thymidine kinase–deficient
Accepted June 28, 2019.
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You are going to email the following IL-17 Production by γδ+ T Cells Is Critical for Inducing Th17 Responses in the Female Genital Tract and Regulated by Estradiol and Microbiota
Varun C. Anipindi, Puja Bagri, Sara E. Dizzell, Rodrigo Jiménez-Saiz, Manel Jordana, Denis P. Snider, Martin R. Stämpfli, Charu Kaushic
ImmunoHorizons July 1, 2019, 3 (7) 317-330; DOI: 10.4049/immunohorizons.1900040
Expression Levels of Lamin A or C Are Critical to Nuclear Maturation, Functional Responses, and Gene Expression Profiles in Differentiating Mouse Neutrophils
Laminin Isoforms in Human Dental Pulp: Lymphatic Vessels Express Laminin-332, and Schwann Cell–Associated Laminin-211 Modulates CD163 Expression of M2-like Macrophages
A Genetic Variation of Lipopolysaccharide Binding Protein Affects the Inflammatory Response and Is Associated with Improved Outcome during Sepsis
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redpajama_set_name: RedPajamaCommonCrawl